Fig. 5.

Comparison of nonspecific dye and LAMP-BEAC performance in detecting SARS-CoV-2 RNA with increased reaction volume. Times to threshold was estimated in reactions amplifying synthetic SARS-CoV-2 RNA diluted in inactivated saliva with the N2 primer set with modified LB primer in total reaction volumes of 10 μl (a–c), 20 μl (d–f), and 200 μl (g–i). Time to threshold was calculated for both nonspecific dye (a, d, g) and molecular beacon N2_LBMB_S3 labeled with a cyanine-3 fluorophore (b, e, h) within each reaction. Dashed horizontal line indicates threshold used to call a well positive or negative; less than 20 min for nonspecific dye in 10 or 20 μl reactions, less than 17 min for nonspecific dye in 200 μl reactions and 80 min (the time the reactions were terminated) for the molecular beacon. For ease of comparison, dots are colored blue if called positive by nonspecific dye and molecular beacon, yellow if called positive by molecular beacon alone, and gray if called negative by both methods (no sample was called positive by nonspecific dye and negative by molecular beacon). Times to threshold for molecular beacon wells with no observed increase in fluorescence were arbitrarily set to 85 min. Points are offset slightly on the x-axis for visualization. g–i A comparison of the proportion of wells called positive in the reaction shown in a–b, d–e, and g–h by nonspecific dye and molecular beacon (blue) or molecular beacon alone (yellow) in 10 μl (g), 20 μl (h), or 200 μl (i) reactions (n=24 for each dilution)